Persistent Spike-specific T cell immunity despite antibody reduction after 3 months from SARS-CoV-2 BNT162b2-mRNA vaccine

Vaccine is the main public health measure to reduce SARS-CoV-2 transmission and hospitalization, and a massive scientific effort worldwide resulted in the rapid development of effective vaccines. This work aimed to define the dynamics and persistence of humoral and cell-mediated immune response in Health Care Workers who received a two-dose BNT162b2-mRNA vaccination. Serological response was evaluated by quantifying anti-RBD and neutralizing antibodies while cell-mediated response was performed by a whole blood test quantifying Th1 cytokines (IFN-γ, TNF-α, IL-2) produced in response to Spike peptides. BNT162b2-mRNA vaccine induced both humoral and cell-mediated immune response against Spike in all HCW early after the second dose. After 12 weeks from vaccination, the titer of anti-RBD antibodies as well as their neutralization function decreased while the Spike-specific T-cells persisted at the same level as soon after vaccine boost. Of note, a correlation between cellular and humoral response persevered, suggesting the persistence of a coordinated immune response. The long lasting cell-mediated immune response after 3 months from vaccination highlight its importance in the maintaining of specific immunity able to expand again to fight eventual new antigen encountering.

Vaccine is the main public health measure to reduce SARS-CoV-2 transmission and hospitalization, and a massive scientific effort worldwide resulted in the rapid development of effective vaccines. This work aimed to define the dynamics and persistence of humoral and cell-mediated immune response in Health Care Workers who received a two-dose BNT162b2-mRNA vaccination. Serological response was evaluated by quantifying anti-RBD and neutralizing antibodies while cell-mediated response was performed by a whole blood test quantifying Th1 cytokines (IFN-γ, TNF-α, IL-2) produced in response to Spike peptides. BNT162b2-mRNA vaccine induced both humoral and cell-mediated immune response against Spike in all HCW early after the second dose. After 12 weeks from vaccination, the titer of anti-RBD antibodies as well as their neutralization function decreased while the Spike-specific T-cells persisted at the same level as soon after vaccine boost. Of note, a correlation between cellular and humoral response persevered, suggesting the persistence of a coordinated immune response. The long lasting cell-mediated immune response after 3 months from vaccination highlight its importance in the maintaining of specific immunity able to expand again to fight eventual new antigen encountering.
Large-scale vaccination is the single most effective public health measure for mitigation of the coronavirus disease (COVID-19) pandemic and a global effort to develop and distribute an effective vaccine produced several effective options.
Several data are currently available about the effectiveness of BNT162b2 (two-dose vaccination regimen administered 21 days apart) in inducing a strong antibody and cell-mediated immune response in healthy individuals with a 95% efficacy in naïve individuals [1][2][3] . Moreover, the coordinate induction of both humoral-and cell-mediated arms has been clearly demonstrated more effective in fighting SARS-CoV-2 infection 4,5 .
The persistence of a specific immune response represents a crucial point to be addressed in order to define the risk of re-infection and to optimize the vaccination strategies. In convalescent patients, several data confirmed that both antibodies and T cell response persisted 6-8 months after the infection 5,6 but no definitive data are available in vaccinated individuals.
In this study, we established the dynamics and persistence of the immune response by assessing the both anti-Region-Binding-Domain (RBD) antibody and Spike (S)-specific T-cell response early and after 12 weeks from vaccination in a cohort of HCWs who received a two-dose BNT162b2-mRNA vaccination at the National Institute for Infectious Diseases L. Spallanzani in Rome. Antibody evaluation and micro-neutralization assay (MNA 90 ). Two commercial chemiluminescence microparticle antibody assays (ARCHITECT ® i2000sr Abbott Diagnostics, Chicago, IL, USA) were used: the anti-Nucleoprotein IgG and the SARS-CoV-2 IgG II kit, detecting antibodies against the RBD of SARS-CoV-2. The neutralization assay was performed according to 7 , using SARS-CoV-2/Human/ITA/PAVIA10734/2020, provided by Fausto Baldanti, Pavia, as challenging virus.
T-cell immune response. Peripheral blood was collected in heparin tubes and stimulated or not with a pool of peptides spanning the Spike protein (Miltenyi Biotech, Germany) at 37 °C (5% CO 2 ), according to [8][9][10] . A superantigen (SEB) was used as positive control. Plasma were harvested after 16-20 h of stimulation and stored at − 80 °C. Th1-cytokines (IFN-γ, TNF-α, IL-2) were quantified in the plasma samples using an automatic ELISA (ELLA, Protein Simple). The detection limit of these assays were 0.17 pg/ml, 0.3 pg/ml and 0.54 pg/ml for IFN-γ, TNF-α and IL-2 respectively.

Statistical analysis.
Continuous variables including anti-RBD, anti-N, MNA 90 titers, IFN-γ, TNF-α, IL-2 levels were reported as median and interquartile range (IQR). Comparison of medians across groups were evaluated by Wilcoxon for pairwise comparisons. Correlations between assays were assessed by non-parametric Spearman's rank tests. Analyses were performed using Graph Pad. A 2-sided P value < 0.05 was considered statistically significant.
We therefore focused on the immune response at T12w in order to define the coordination of both humoral and cell-mediated immunity (Fig. 1C). A positive correlation between anti-RBD and MNA90 was maintained (r = 0.5606, p < 0.0001), although decrease respect to that observed early after vaccination (r = 0.8103, p < 0.0001). The S-specific T cell response at T12w was characterized by the production by Th1 cytokines and the amount of IFN-γ correlated with IL-2 (r = 0.6733, p < 0.0001) and TNF-α (r = 0.6733, p < 0.0001). Finally, a slight but significant correlation between anti-RBD antibody and IFN-γ (R = 0.3421, p < 0.0001), IL-2 (r = 0.3441, p < 0.0001) and TNF-α (R = 0.2735, p < 0.0001) has been reported, suggesting the persistence of a coordinated humoral and cell-mediated immune response.

Discussion
The vaccination strategies are aimed to induce a protective immunity with persistent humoral and cell-mediated immune response. In this context, Yellow Fever and smallpox vaccines represent good examples inducing remarkably effective and long-lived immune protection through the induction of neutralizing antibodies and T-cells with broad specificity, high magnitude, polyfunctionality, high proliferative potential and long-term persistence 11 .
In this study, we described the persistence of both humoral and cell-mediated immune response to Spike in HCW after 12 weeks from vaccination. Nevertheless, while anti-RBD and neutralizing titer decreased overtime, the S-specific T cell immunity persisted at similar level.   www.nature.com/scientificreports/ The ability of BNT162b2 mRNA to induce a strong humoral and cell-mediated immune response early after vaccination was confirmed in several studies 1-3 but data on their persistence have not still clarified. In convalescent SARS-CoV-2 patients growing evidences support the persistence of the immune response more than 8 months both in symptomatic and in asymptomatic patients 5,12,13 . During the acute phase of infection, the induction of a coordinate immune response, by neutralizing antibodies production and specific T-cell response resulted effective against the virus and represents a main feature of mild disease 4,14 . After recovery, each component of SARSCoV-2 immune memory persisted and differentiate in memory cells 5 .
Here we showed that two dose of BNT162b2 mRNA vaccine in healthy subjects induced a strong anti-RBD and neutralizing antibody response which however begin to decrease after 12 weeks from vaccination prime. The decrease of effector antibody-producing B cells could be associated to the differentiation of the specific memory compartment, as demonstrated after natural infection 5 . Interestingly, despite the antibody reduction, a persistent T cell immunity has been observed and S-specific T lymphocytes produced IFN-γ and IL-2 at similar extent as early after vaccination, suggesting the maintenance of a robust pool of effector Ag specific T cells until 12 weeks. The ability to respond to specific stimulation producing several Th1 cytokines may represent a good marker of protective phenotype as shown in convalescent COVID-19 patients 13 . In particular, the production of IL-2 can be essential for the homeostatic maintenance of a functional specific T-cell response. Moreover, we found a positive correlation between the two arms of the immune system that can be important to generate a balanced memory pool of B and T lymphocytes. A reduced level of neutralizing antibody at 6 months post-vaccination as well as memory B and T cells was reported 15 . Of note, vaccine-induced memory B cells can recognize the variants of concern showing a high hypermutation in variant-binding B cell clones, suggesting a continued evolution to fight new variants.
Overall, our study describes the different kinetic of anti-RBD and Cell-mediated immune response after 12 weeks from BNT162b2 mRNA vaccine in a cohort of HCWs. Despite a significant decrease of antibody, the specific T cells able to produce several Th1 cytokines persisted as early after booster dose to elicit a coordinated neutralizing humoral and cytokine producing S-specific T-cell response in HCW. The study has been conducted in a single center and recruited healthy, young and middle aged adults, predominantly female, without previous SARS-CoV-2 infection, and its findings should not be generalized to different population.
Assessing the dynamics of these parameters is essential to evaluate the long-term protection and to establish its duration. These results will be crucial to generate the evidence needed for a better understanding of the optimal vaccination strategy.

Data availability
The data presented in this study are available on request from the corresponding author.